After a patient study successfully demonstrated the use of exhaled limonene as a breath biomarker to measure liver function and stage liver disease, Owlstone Medical explains more about its EVOC Probes.
Figure 1: The EVOC Probe strategy for biomarker development. Administered EVOC Probes allow non-invasive monitoring of target disease and metabolic processes through breath sample collection and analysis using Breath Biopsy.
Typical biomarker discovery can feel like panning for gold, sifting through hundreds of possible factors captured from a specific method in the hope of finding ‘the one’ with clinical potential. But there is another way. Chemicals known as exogenous volatile organic compound probes (EVOC Probes), are safe compounds that can be administered to assess targeted metabolic or disease processes, comparable to the molecular probes used in PET scans.
Since EVOC Probes aren’t found within the body itself, they provide a high contrast signal for detection, and each Probe is developed on the basis of clear biological connections to the process of interest. Analysis methods can also be optimised with the EVOC Probe to further enhance accurate, high-sensitivity detection. What’s more, EVOC Probes work in conjunction with Breath Biopsy, a non-invasive platform that reproducibly collects and analyses biomarkers from exhaled breath.
A new paper published in Clinical and Translational Gastroenterology demonstrates a potential EVOC Probe, in this case limonene, with possible applications for the detection, staging and progression tracking of liver disease. A connection between limonene and liver diseases was first shown by Chris Mayhew and colleagues at the University of Birmingham who found that the levels of exhaled limonene on breath fall following liver transplants. In the new paper, Ferrandino et al. focus on cirrhosis patients, but the same approach could be suitable for application to early stage diseases such as non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH), offering a powerful tool for early disease detection.
The study collected breath and blood samples from 84 subjects including healthy volunteers and cirrhosis patients either with or without hepatocellular carcinoma. Targeted analysis of the breath samples using Breath Biopsy revealed that levels of limonene in samples were higher in cirrhosis patients, especially in those with more severe disease (Child-Pugh classification). A predictive model based on the data was 75% accurate (73% sensitivity, 77% specificity) based on a threshold of 10.2 ng/L of limonene on breath.
Figure 2: Measurements of exhaled limonene on the breath of test subjects represented as boxplots. Left – All subjects grouped by disease status. Right – Cirrhosis patients stratified by Child-Pugh class, a measure of disease severity.
The study goes on to compare limonene measurements on breath to known blood indicators of liver function and damage. The results show significant correlations with three other liver function markers (bilirubin, albumin and international normalised ratio (INR)) and no correlation with alanine aminotransferase (ALT), a measure of liver damage. This indicates that limonene has great potential to detect early stage liver diseases where function may be mildly affected but liver damage is minimal. The strengths of the EVOC Probe approach make it well suited for early disease detection where disease-associated perturbations are more subtle.
The goal of this work is to identify a robust panel of biomarkers for liver diseases with the aim of developing a widely-available test for early stage disease detection and progression monitoring. Globally, factors such as poor diet, obesity and type-II diabetes are contributing to a rapid rise in the prevalence of these diseases, with as many as 3 in 10 adults and 1 in 10 children affected. Currently many cases are being missed because the early stages are largely asymptomatic. The hope is that a Breath Biopsy test for liver disease will allow more people to take action before they cause irreversible damage to their livers, improving public health and reducing the burden on healthcare systems.
Figure 3: Left – A receiver operator characteristic (ROC) curve illustrating the capability to apply a limonene threshold to separate healthy controls from cirrhosis patients. The optimal result is achieved for a threshold of 10.2 ng/L of limonene on breath. Right – Relating limonene levels to other liver disease biomarkers identified from blood testing. Albumin, bilirubin and INR are indicators of liver function, while ALT reflects liver damage.
A key strength of the Breath Biopsy method used in this study is that it enables simultaneous targeted and untargeted analysis of the same breath samples. The recent publication includes only the targeted analysis but the untargeted results – that aim to discover new biomarkers – are forthcoming. Follow-up studies will aim to test the EVOC Probe method in practice, administering a standard dose of limonene to each subject before breath collection, and will seek to recruit larger cohorts encompassing a wider range of liver disease stages.
Breath Biopsy has many applications beyond liver disease and is available for integration into research studies across a wide range of conditions and applications including respiratory diseases, cancer and gut health.
